The overall objective of the project is a fundamental understanding of the biologically important chemistry of lipid-derived endoperoxides and the products generated from these key intermediates in oxidative transformations of polyunsaturated fatty acids. Protein modifications by lipid-derived electrophiles are gaining recognition as biologically important reactions. A well-known example is the oxidative conversion of LDLs into oxLDL that is associated with the development of atherosclerotic plaques. We hypothesize that a diverse family of protein modifications are generated by condensations with levulinaldehydes derived from lipid endoperoxides. For example, secoprostanoic acid levulinaldehyde derivatives, levuglandins (LGs), are generated from the endoperoxide, PGH2. Furthermore, LGs are rapidly sequestered by proteins forming adducts that are often themselves highly chemically reactive. Progress achieved in the past two years includes: 1) the preparation of several specific polyclonal antibodies that provide sensitive reagents for the detection of LGE2-derived protein-bound pyrroles; 2) the demonstration in vitro that protein-bound LG-derived pyrroles are unexpectedly stable; 3) the discovery that LG-pyrrole immunoreactivity is produced during in vitro oxidation of LDLs; 4) the detection in vivo of disease-related levels of LG-pyrrole immunoreactivity in tissues and blood plasma; 5) the discovery that an LG-LDL adduct inhibits the hydrolase activity of cathepsin B, a lysosomal enzyme found in macrophages. In spite of the progress achieved in our laboratory and by our collaborators, the discoveries listed above raise more questions than they answer. First and foremost, the structures of the novel antigens that we have detected in vivo must be thoroughly characterized and the intermediacy of LGE2 derivatives in their formation from LDLs must be confirmed. Moreover, it is likely that besides the levuglandins, six as yet undetected isomers of LGs, isoLGs, are generated during oxidation of LDLs. Therefore, in the coming three years, we propose to: 1) isolate and characterize stable derivatives of the modified amino acids in LG-protein adducts; 2) trap and characterize LG intermediates produced during oxidation of LDLs; 3) prepare isoLGs by total synthesis; 4) prepare immunological and isotopically labeled analytical tools for LG and isoLG- protein adducts; 5) characterize the structures, levels, and biologically important chemistry of all LG and isoLG-derived protein adducts present in plasma from coronary artery bypass, renal failure, stroke, and heart attack patients. By the end of this funding period we hope to have elucidated the major chemical pathways for the formation and reactions of protein adducts of arachidonic acid-derived levulinaldehydes under physiological conditions as a basis for in-depth studies of their biological significance.